Expression of heat shock protein70 in pig oocytes: Heat shock response during oocyte growth

Animal Reproduction Science 96 (2006) 154–164

Expression of heat shock protein70 in pig oocytes: Heat shock response during oocyte growth Vilma L´ansk´a a,∗ , Eva Chmel´ıkov´a a , Mark´eta Sedm´ıkov´a a , Jaroslav Petr b , Radko Rajmon a , Michal Jeˇseta a , Jiˇr´ı Rozinek a a

Czech University of Agriculture in Prague, Department of Veterinary Sciences, Kam´yck´a 129, 165 21 Prague 6-Suchdol, Czech Republic b Research Institute of Animal Production, Pˇra ´ telstv´ı 815, Prague 10-Uhˇr´ınˇeves, Czech Republic

Received 24 June 2005; received in revised form 16 November 2005; accepted 16 December 2005 Available online 18 January 2006

Abstract The heat shock response of growing and fully-grown pig oocytes was analyzed in vitro by determining heat shock protein70 (HSP70) synthesis under both normal conditions (39 ◦ C; 0 and 6 h) and after heat shock (43 ◦ C; 1, 4 and 6 h). The expression of HSP70 in oocytes was detected by immunoblotting analysis. Growing oocytes measuring 80–99 ␮m synthesized a high number of HSP70 without heat shock effect, and these were capable of increasing the synthesis of HSP70 after heat shock to a maximum after 1 h. Growing oocytes measuring 100–115 ␮m also synthesized HSP70 without heat shock and after it, but the HSP70 synthesis was not statistically changed by increasing duration of heat shock. In fully-grown oocytes, great amounts of HSP70 were found without heat shock treatment, and the contents of HSP70 significantly decreased after heat shock. These results indicate that growing oocytes are able to synthesize HSP70 after heat shock. This ability declines at the end of the growth period, and fullygrown oocytes are unable to induce HSP70 synthesis after heat shock. HSP70 is synthesized and stored during oocyte growth. The high HSP70 synthesis in non-heat-treated growing oocytes and a great amount of HSP70 in fully-grown oocytes support the hypothesis that HSP70 is important for oocyte growth and maturation. © 2006 Elsevier B.V. All rights reserved. Keywords: Heat shock protein; Oocyte; Pig; Western blotting

∗

Corresponding author. Tel.: +420 224382952; fax: +420 23438141. E-mail address: [email protected] (V. L´ansk´a).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2005.12.005

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1. Introduction It has been well documented that maturing oocytes are very susceptible to heat stress as well as to other environmental stress factors. Direct exposure of maturing oocytes to elevated temperatures disrupts spindle formation during metaphase I in mice (Baumgartner and Chrisman, 1981), reduces the frequency of oocytes that progress to metaphase II, and decreases the rate of fertilization in mice and cattle (Lenz et al., 1983; Baumgartner and Chrisman, 1981). Abnormalities in the chromosomes and cytoskeleton after heat shock were also observed in matured pig oocytes (Ju and Tseng, 2004). In addition, Tong et al. (2004) demonstrated that transient exposure to elevated temperature during postslaughter processing caused extensive cytoskeletal damage, which in turn, drastically decreased developmental competence. The heat sensitivity of mammalian oocytes indirectly suggests that oocytes might not be equipped with proper mechanisms for heat-stress protection. Previous results support the hypothesis that the high sensitivity of oocytes during meiotic maturation is related to the absence of inducible synthesis of heat shock protein70 (HSP70) (Neuer et al., 1999), a key molecule in the protection of cells from heat shock and other stresses (Ananthan et al., 1986; Lindquist, 1986; Welch, 1992; Hansen, 1999). Moreover, microinjection of HSP70 mRNA to oocytes enhanced resistance to heat shock (Hendrey and Kola, 1991). In mice, Curci et al. (1987) ascertained that oocytes at the preovulatory stage of their differentiation lack an inducible synthesis of heat shock proteins and display a high sensitivity to hyperthermia. Growing mouse oocytes, in contrast to preovulatory oocytes, exhibit a heat shock response represented by a heat-elicited synthesis of HSP70 isoform (designated as HSP68 by Curci et al., 1991). The respondence of fully-grown oocytes to hyperthermia declines during antral follicle development and is finally arrested with oocyte/follicle terminal differentiation (Curci et al., 1987, 1991). Fully-grown oocytes from preovulatory follicles are incapable of responding to heat shock due to synthesis of the HSP70 inducible form, but contain stored constitutive (HSC70) and inducible (HSP70) isoforms of the HSP70 protein family, as demonstrated by Liu et al. (2004). Not only were the HSC70 and HSP70 proteins found stored in GV stage mouse oocytes, but they also displayed a higher concentration than did metaphase II (M II) stages of mouse oocytes and embryos before implantation. The inability to induce heat shock protein synthesis after heat shock was also confirmed in bovine oocytes. Immature bovine oocytes can synthesize two HSC70 molecules and protein corresponding to inducible HSP70. However, the rate of synthesis of these proteins is not altered by heat shock (Edwards and Hansen, 1996). Following oocyte maturation, synthesis of one of the HSC70 proteins ceases, and the rates of synthesis of the other two heat shock proteins 70 remain unaffected by heat shock (Edwards and Hansen, 1997; Edwards et al., 1997). The distribution of HSP70 in the ooplasm of immature and mature oocytes is also unaffected by exposure to elevated temperatures, and this protein was closely associated with the meiotic spindle, indicating its possible role in stabilizing this structure (Kawarsky and King, 2001). These cited findings suggest that HSP70 is important for oocyte growth and maturation, and document a block of heat induction of HSP70 during oocyte maturation. In this study, we extended our knowledge of the capacity of pig oocytes to synthesize HSP70 in response to elevated temperature because, the expression of HSP70 in oocytes has not yet been determined in the pig. Therefore, HSP70 was identified in the one-cell stage of pig embryos (King et al., 2000), which supports the hypothesis that these stress proteins are part of maternal resources because activation of the embryonic genom in pigs occurs later, at the four-cell stage. The purpose of our study was to verify the hypothesis that fully-grown pig oocytes are unable to increase synthesis of the heat inducible HSP70 form after heat shock and that they

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contain stored maternal heat shock proteins HSP70 which are synthesized during the growth phase of oogenesis. We also investigated whether exposure of growing and fully-grown oocytes (GV stage) to elevated temperatures during postslaughter processing could affect their HSP70 synthesis. 2. Materials and methods 2.1. Oocyte collection Pig ovaries were obtained from a local slaughterhouse from gilts at an unknown phase of the oestrous cycle and transported to the laboratory within 2 h in a saline solution (0.9% sodium chloride) at 39 ◦ C. The fully-grown oocytes (i.d. 120–125 ␮m excluding zona pellucida) were collected by aspiration of follicles of 2–5 mm in diameter with a 20-gauge needle. The growing oocytes (80–99 and 100–115 ␮m in internal diameter) were obtained from thin strips (2–3 mm width, 10–15 mm length) dissected from the surfaces of the ovaries. The strips were placed in Petri dishes containing culture medium. The growing oocytes were liberated from their follicles by opening the follicular wall using the needle. After isolation, the fully-grown and growing oocytes were denuded of their surrounding follicle cells by mechanical pipetting with a small-bore pipette. The diameters of oocytes were measured with an ocular micrometer. The oocytes were cultured and exposed to heat shock in a modified M199 medium (GibcoBRL, Life Technologies, Paisley, Scotland) containing sodium bicarbonate (0.039 mL of a 7.0% solution per milliliter of the medium), calcium lactate (0.6 mg/mL), gentamicin (0.025 mg/mL), HEPES (1.5 mg/mL) and 10% of fetal calf serum (GibcoBRL, Life Technologies, Germany, Lot No. 40F2190F). The oocytes were cultured in 3.5-cm diameter Petri dishes (Nunc, Roskilde, Denmark) containing 3.0 mL of the culture medium. 2.2. Heat shock treatments and oocyte extract preparation Both groups of growing oocytes and fully-grown oocytes were exposed to heat shock at 43 ◦ C for 1, 4 and 6 h, in Experiments 1–3, in a humidified atmosphere of 5% CO2 in air. Control (nonheat-treated) growing and fully-grown oocytes were cultured for 0 and 6 h at 39 ◦ C in a mixture of air with 5% CO2 and 100% humidity in Experiments 1–3. Both groups of growing oocytes and fully-grown oocytes were then washed three times in a culture medium and then in Tris buffer saline (TBS) and transferred in a minimum volume of TBS to 50 ␮L of SDS lysis buffer. Each sample was prepared from 20 fully-grown or growing oocytes. Proteins from oocytes were precipitated by trichloracetic acid. The samples were centrifuged for 5 min at 10,000 × g and the supernatant was removed. The samples were frozen and stored at −20 ◦ C until processed for gel electrophoresis. 2.3. Immunoblotting analysis Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein samples were diluted in a 20 ␮L SDS sample buffer. Two ng of purified protein HSP70 (Stressgen Biotechnologies, Canada) per well were run simultaneously as a positive control. Following separation, the proteins were electrophoretically transferred to nitrocellulose membranes. Successful transfer of the proteins was verified by the transfer of the prestained molecular

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weight standards (Prestained SDS-PAGE Standards, Bio-Rad Lab., Canada). The membranes were then placed in TBS-T (2 M Tris–HCl, pH 7.5, 4 M NaCl, 0.1% Tween-20) containing 5% non-fat dry milk for 90 min at room temperature. The membranes were washed in TBS-T and then incubated overnight with a mouse anti-HSP70 monoclonal antibody (SPA-810; StressGen Biotechnologies Corp., BC, Canada) diluted 1:20,000 in TBS-T at 4 ◦ C. After TBS-T washes, the membranes were reacted with secondary anti-mouse antibody conjugated with horseradish peroxidase (diluted 1:120,000 in TBS-T, Amersham Biosciences Corp., UK) at room temperature for 90 min. The membranes were again rinsed with TBS-T. Peroxidase activity was detected by chemilluminiscence using the ECL Advance Western Blotting Detection Kit (Amersham Biosciences Corp.). The amount of HSP70 in each spot was measured with an image analyzing system (L.U.C.I.A, v. 4.71, Laboratory Imaging, s.r.o.) by performing densitometric analysis. The content of HSP70 was measured on the basis of the integrated optical density (IOD) parameter (mean optical density × area of induction). The same method was used, e.g. by Vogel et al. (1997) for measuring the amount of synthesized HSP70. For comparison of HSP70 quantities among the blots, the values of IOD were related to values of IOD of purified HSP70 protein. The purified HSP70 protein was used in the same amount—2 ng-in every blot. The values of relative integrated optical density (RIOD) are presented as the mean with ± standard deviation. 2.4. Experimental design In Experiment 1, we investigated the heat shock response of growing oocytes 80–99 ␮m in internal diameter. Denuded oocytes were cultured in vitro at 43 ◦ C for 1, 4 and 6 h. Control oocytes were cultured without cumulus cells at 39 ◦ C, 0 and 6 h. Experiment 2 was performed to investigate the heat shock response of growing oocytes 100–115 ␮m in internal diameter. Denuded oocytes were cultured in vitro at 43 ◦ C for 1, 4 and 6 h. Control oocytes were cultured without cumulus cells at 39 ◦ C, 0 and 6 h. In Experiment 3, we investigated the heat shock response of fully-grown oocytes 120–125 ␮m in internal diameter and their stored amount of HSP70. Denuded oocytes were cultured in vitro at 43 ◦ C for 1, 4 and 6 h. Control oocytes were cultured without cumulus cells at 39 ◦ C, 0 and 6 h. One hundred oocytes were analyzed in one experiment (five samples consisting of 20 oocytes each). The Experiments 1–3 were repeated four times, the total number of used oocytes was 400 in each experiment. Experiment 4 was performed to elucidate the influence of elevated temperature during postslaughter processing on HSP70 synthesis in oocytes. Gilts were stunned by electrical shock and subsequently bled off. Immediately after slaughter, before scalding, the ovaries were taken away by surgical intervention as control. Only one ovary was obtained from each gilt (n = 4), and the abdominal cavity was closed by surgical suture. The gilts were then scalded for dehairing in a scalding tub at 65 ◦ C for 10 min. After scalding, the other ovary was removed. In this experiment, the amount of HSP70 in both categories of growing oocytes (80–99, 100–115 ␮m) and fully-grown oocytes (120–125 ␮m) from ovaries obtained before (un-scalded) and after scalding (scalded) of gilts was compared. The temperature in the abdominal cavity was measured during the taking of ovaries before and after scalding. The mean value of temperature in the abdominal cavity was 39.5 ± 0.5 ◦ C before scalding immediately after slaughter and 41.0 ± 0.5 ◦ C when the temperature was measured after scalding. A total of 120 oocytes were analyzed in one experiment (six samples consisting of 20 oocytes each). The Experiment 4 was repeated four times. The total number of used oocytes was 480.

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2.5. Statistical analysis Data from all experiments were subjected to statistical analysis. The experiments were repeated four times. The data were analyzed by least square analysis of variance using the program Statgraphics v.5. The differences among the groups were determined by the Duncan method. P-values less than 0.05 were considered statistically significant. 3. Results 3.1. Expression of HSP70 during oocyte growth Heat shock proteins HSP70 were detected in growing oocytes measuring 80–99 ␮m (Fig. 1). These oocytes synthesized a high amount of the inducible form HSP70 without heat shock effect. In these oocytes the contents of inducible HSP70 were completely depleted during 6-h cultivation at a temperature of 39 ◦ C. After heat shock of 43 ◦ C, the oocytes were capable of increasing synthesis of HSP70 with a maximum of HSP70 expression after 1-h heat shock treatment (Fig. 2). Following a longer, 4-h heat shock treatment, the contents of these stress proteins markedly decreased and, after 6-h heat shock treatments, HSP70 synthesis significantly increased.

Fig. 1. Western blot analysis of HSP70 expression in growing oocytes of pigs measuring 80–99 ␮m. Lane M represents the molecular weight standards. Lane 1 represents 2 ng of purified HSP70 protein. Lanes 2 and 3 represent control groups, 39 ◦ C for 0 h and 39 ◦ C for 6 h, respectively. Lanes 4–6 represent heat-shocked groups, 43 ◦ C, 1 h; 43 ◦ C, 4 h; 43 ◦ C, 6 h, respectively. Each lane contains protein samples from 20 oocytes.

Fig. 2. Bar graph for relative integrated optical density of HSP70 expression for control (39 ◦ C, 0 and 6 h) and heat-shocked (43 ◦ C, 1, 4 and 6 h) groups from growing pig oocytes 80–99 ␮m in diameter. The HSP70 contents were determined according to their integrated optical density (IOD) related to IOD of purified HSP70 protein. The different letters (a–e) denote statistically significant differences in the RIOD of HSP70 expression (P < 0.05).

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Fig. 3. Western blot analysis of HSP70 expression in growing oocytes of pigs measuring 100–115 ␮m. Lane M represents the molecular weight standards. Lane 1 represents 2 ng of purified HSP70 protein. Lanes 2 and 3 represent control groups, 39 ◦ C for 0 h and 39 ◦ C for 6 h, respectively. Lanes 4–6 represent heat-shocked groups, 43 ◦ C, 1 h; 43 ◦ C, 4 h; 43 ◦ C, 6 h, respectively. Each lane contains protein samples from 20 oocytes.

As shown in Fig. 3, growing oocytes measuring 100–115 ␮m synthesized HSP70, both without heat shock treatment and after it. This category of growing oocytes contained a significantly lower amount of HSP70 after heat shock, independent of its duration, than the oocytes without heat shock treatment immediately after isolation from the follicles or after 6-h cultivation at 39 ◦ C (Fig. 4). HSP70 synthesis was not statistically changed with increasing duration of the heat shock. 3.2. Expression of HSP70 after termination of oocyte growth A great amount of stored stress proteins was found in fully-grown oocytes (Fig. 5). The contents of HSP70 significantly decreased after cultivation at 39 ◦ C for 6 h (Fig. 6). Fully-grown oocytes depleted approximately one-third of their store of HSP70 during the 6-h cultivation when cumulus cells were removed before the cultivation. The lack of stores of HSP70 following heat shock in fully-grown oocytes was significantly high. After 1-h heat shock, a low level of HSP70 was still present, but after 4 and 6 h, it was completely absent.

Fig. 4. Bar graph for relative integrated optical density of HSP70 expression for control (39 ◦ C, 0 and 6 h) and heat-shocked (43 ◦ C, 1, 4 and 6 h) groups from growing pig oocytes 100–115 ␮m in diameter. The HSP70 contents were determined according to their integrated optical density (IOD) related to IOD of purified HSP70 protein. The different letters (a–c) denote statistically significant differences in the RIOD of HSP70 expression (P < 0.05).

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Fig. 5. Western blot analysis of HSP70 expression in fully-grown pig oocytes (GV stage). Lane M represents the molecular weight standards. Lane 1 represents 2 ng of purified HSP70 protein. Lanes 2 and 3 represent control groups, 39 ◦ C for 0 h and 39 ◦ C for 6 h, respectively. Lanes 4–6 represent heat-shocked groups, 43 ◦ C, 1 h; 43 ◦ C, 4 h; 43 ◦ C, 6 h, respectively. Each lane contains protein samples from 20 oocytes.

Fig. 6. Bar graph for relative integrated optical density of HSP70 expression for control (39 ◦ C, 0 and 6 h) and heatshocked (43 ◦ C, 1, 4 and 6 h) groups from fully-grown pig oocytes (GV stage). The HSP70 contents were determined according to their integrated optical density (IOD) related to IOD of purified HSP70 protein. The different letters (a–d) denote statistically significant differences in the RIOD of HSP70 expression (P < 0.05).

3.3. Effect of elevated temperatures during the slaughter procedure on HSP70 expression in oocytes Expression of HSP70 was detected in the un-scalded and scalded growing oocytes measuring 80–99 ␮m (Fig. 7), and the amounts of this protein were significantly different—3.13 ± 0.20 ng

Fig. 7. Western blot analysis of HSP70 expression in un-scalded and scalded growing and fully-grown pig oocytes during slaughter procedure. Lanes 1–3 represent the purified HSP70 protein (1, 2, 5 ng, respectively). Lanes 4 and 5 represent un-scalded and scalded growing pig oocytes measuring 80–99 ␮m, respectively. Lanes 6 and 7 represent un-scalded and scalded growing pig oocytes measuring 100–115 ␮m, respectively. Lanes 8 and 9 represent un-scalded and scalded fully-grown pig oocytes (GV), respectively.

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versus 4.15 ± 0.35 ng (in 20 oocytes). HSP70 was also detected in growing oocytes measuring 100–115 ␮m, both without exposure to elevated temperature during postslaughter processing and also after heating-up during scalding (Fig. 7), 4.05 ± 0.15 ng and 3.25 ± 0.26 ng HSP70 (in 20 oocytes), respectively. HSP70 synthesis in un-scalded growing oocytes (100–115 ␮m) was markedly higher than in oocytes obtained after scalding. The HSP70 content in fully-grown oocytes was 4.10 ± 0.19 ng in 20 oocytes that were un-scalded and 2.55 ± 0.45 ng in 20 oocytes isolated from ovaries after scalding (Fig. 7). The synthesis of HSP70 in both categories of unscalded growing oocytes and un-scabled fully-grown oocytes was not induced during scalding of the animals in the slaughterhouse. 4. Discussion We demonstrated the presence of HSP70 in growing and fully-grown pig oocytes. HSP70 was detected in both groups of growing pig oocytes immediately after their isolation from follicles, without exposure to heat shock. The results of Experiments 1 and 2 confirm that the inducible HSP70 isoform is synthesized during the growth of oocytes under normal conditions. This situation in pigs is different from that in mice, as documented by Curci et al. (1991), who did not demonstrate HSP70 expression in growing mouse oocytes under physiological conditions. We therefore studied whether HSP70 expression was not induced by the increased body temperature resulting from the scalding of pigs after slaughter. The differences in HSP70 contents between the oocytes isolated before scalding and those isolated after scalding were significant in both size categories of growing oocytes. In spite of the fact that the increase in body temperature during the scalding of pigs affects HSP70 expression, it was demonstrated that growing oocytes synthesize HSP70 under physiological conditions without heat shock. The synthesis of stress proteins in growing oocytes without heat shock treatment seems to be related to their high synthesizing activity (Hyttel et al., 2001) and we assumed that the high level of inducible HSP70 is related to the necessary protection of newly synthesized proteins (Beckmann et al., 1990; Fink, 1999). The cultivation itself, together with the removal of cumulus cells, represents relatively high stress for both groups of growing oocytes, and they respond to it by depletion of HSP70 (80–99 ␮m) or induction of HSP70 synthesis (100–115 ␮m). As is evident from the results obtained by Christians et al. (1995), the culture conditions themselves may become a stressing factor. The authors recorded 5–15 times higher expression of gene transcripts of HSP70 in two-cell stages of mouse embryos cultivated in vitro than in the same stage of embryos developed in vivo. After heat shock of 43 ◦ C, the expression of HSP70 in growing oocytes was detected. HSP70 synthesis after heat shock depended on the sizes and development of oocytes during the growth phase. In somatic cells, Gabriel et al. (1996) observed that HSP70 expression was directly dependent on the duration of heat shock treatment. In contrast to the conclusions of the above authors, the stress response by inducing HSP70 synthesis in both groups of growing oocytes had a different course in our experiments. In oocytes measuring 80–99 ␮m, maximum HSP70 expression occurred after 1-h heat shock treatment. Following a longer, 4-h heat shock treatment, the contents of these stress proteins markedly decreased, and after 6-h heat shock, HSP70 synthesis significantly increased. It may be assumed that maximum HSP70 expression occurring as early as 1-h after heat shock treatment, shows the adaptability of growing oocytes (80–99 ␮m) to unfavourable conditions of the environment. On the other hand, growing oocytes measuring 100–115 ␮m contained a significantly lower amount of HSP70 after heat shock, independent of its duration, than oocytes without heat shock

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treatment, immediately after isolation from the follicles. HSP70 synthesis was not statistically changed with increasing duration of heat shock. Towards the end of oocyte growth their transcription and translation activities gradually cease. The oocytes do not synthesize such great amounts of nascent polypeptides, but they contain a large amount of maturated stored proteins (Wassarman, 1988). We therefore assume that the stress response is not as quick and intensive as in the oocytes measuring 80–99 ␮m. In murine oocytes, the heat shock response is also highest during the growth phase of the oocyte and gradually decreases until its final size is reached (Curci et al., 1991). A great amount of stored stress proteins was found in fully-grown oocytes. Our results correspond to the data reported by Liu et al. (2004). These authors also demonstrated a high level of inducible HSP70 in mouse oocytes at the germinal vesicle stage (GV) under physiological conditions. The inducible isoform HSP70 is also present in immature and mature bovine oocytes under physiological conditions (Edwards and Hansen, 1996, 1997; Edwards et al., 1997). Synthesis of stress proteins HSP70 was not induced during scalding of the animals in the slaughterhouse. On the contrary, due to the increase in body temperature during scalding, a part of the HSP70 content was depleted. The depletion of stored HSP70 by transient exposure to elevated temperature during slaughter may be a partial cause of the decrease in developmental competence. Tong et al. (2004) documented that exposure of GV stage pig oocytes to elevated temperatures during postslaughter processing did not have any detrimental effects on nuclear maturation per se, but did result in extensive cytoskeletal damage, which, in turn, drastically decreased developmental competence. However, that was not a problem under our conditions because the embryos developed to the morula or the blastocyst stages during parthenogenetic activation of in vitro maturated oocytes harvested from slaughterhouse ovaries (Petrov´a et al., 2005). The contents of HSP70 significantly decreased after cultivation at 39 ◦ C for 6 h. Fully-grown oocytes depleted approximately one-third of their stored HSP70 during the 6-h cultivation when cumulus cells were removed before cultivation. The loss of stored HSP70 following heat shock in fully-grown oocytes was significant. After 1-h heat shock, a low level of HSP70 was still present, but after 4 and 6 h, it was completely absent. Having terminated their growth, the oocytes entered the stage of meiotic maturation and were transcriptionally inactive (Schultz and Heyner, 1992). Due to this fact, as well as in accordance with our results, it may be assumed that the stress protein HSP70, which is present in the oocytes after termination of their growth, was synthesized during the growth of the oocytes. This is evident not only from the response of fully-grown oocytes to heat shock, but also from their response to increased temperature during scalding and to culture conditions, when the reserve of HSP70 disappeared. HSP70 protein was detected in fully-grown pig oocytes as well as in bovine oocytes (Edwards and Hansen, 1996, 1997; Edwards et al., 1997) and in mouse oocytes after termination of growth (Curci et al., 1991), but no increase in its expression was observed after heat shock treatment. Heikkila et al. (1985) consider this block of heat shock inducibility as a general feature of oogenesis, and our results support this hypothesis. 5. Conclusion On the basis of our results, it is evident that inductive isoforms of HSP70 in growing oocytes are synthesized and maintained at the basal level without stress effect and that they are important for the survival of the oocytes during the growth period. Heat shock induces an increase in the synthesis of HSP70, which is evident from the ability of growing oocytes to synthesize inducible HSP70. This supports our assumption that these stress proteins are synthesized and stored during

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